Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China.
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China.
J Biotechnol. 2024 Sep 20;393:109-116. doi: 10.1016/j.jbiotec.2024.08.001. Epub 2024 Aug 3.
Ganoderma has received much attention for its medicinal value, but the manipulation of multiple genes remains a challenge, hindering the genetic engineering of this species for the development of cell factories. Here, we first showed that the presence of an intron is necessary for the efficient expression of the endogenous cDNA of carboxin-resistant gene (cbx) in G. lucidum. Then, the self-cleaving function of 2 A peptide was investigated in G. lucidum by linking cbx cDNA to the codon-optimized hygromycin B-resistant gene (ophph) using the 2A-peptide sequence. The results showed that cbx cDNA and ophph can be successfully expressed in G. lucidum in a bicistronic manner from a single transcript. Moreover, the expression of both genes was not affected by the order within the 2 A cassette. In addition, simultaneous expression of cbx cDNA, ophph, and codon-optimized yellow fluorescent protein gene (opyfp) was conducted for the first time in G. lucidum using the 2 A peptide-based approach. The developed method was successfully applied to express both cDNA of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (hmgr) and squalene epoxidase gene (se) for enhanced production of ganoderic acids (GAs) in G. lucidum. The engineered strain produced the maximum content of GA-Mk, GA-T, GA-S, and GA-Me were 26.56±3.53,39.58±3.75, 16.54±2.16, and 19.1±1.87 μg/100 mg dry weight, respectively. These values were 3.85-, 4.74-, 3.65-, and 3.23-fold higher than those produced by the control strain. The developed method will be useful for the manipulation of complex metabolic or regulatory pathways involving multiple genes in Ganoderma.
灵芝因其药用价值而备受关注，但多个基因的操纵仍然是一个挑战，这阻碍了该物种的遗传工程，使其无法成为细胞工厂。在这里，我们首先表明内含子的存在对于灵芝中卡巴辛抗性基因（cbx）内源性 cDNA 的高效表达是必要的。然后，通过使用 2A 肽序列将 cbx cDNA 与密码子优化的潮霉素 B 抗性基因（ophph）连接，研究了 2A 肽在灵芝中的自我切割功能。结果表明，cbx cDNA 和 ophph 可以在灵芝中以双顺反子的方式从单个转录本中成功表达。此外，两个基因的表达不受 2A 盒内的顺序影响。此外，首次在灵芝中使用基于 2A 肽的方法同时表达 cbx cDNA、ophph 和密码子优化的黄色荧光蛋白基因（opyfp）。该方法成功地应用于表达灵芝 3-羟基-3-甲基戊二酰辅酶 A 还原酶（hmgr）和角鲨烯环氧化酶基因（se）的 cDNA，以提高灵芝中灵芝酸（GA）的产量。工程菌株产生的 GA-Mk、GA-T、GA-S 和 GA-Me 的最大含量分别为 26.56±3.53、39.58±3.75、16.54±2.16 和 19.1±1.87μg/100mg 干重，分别比对照菌株高 3.85、4.74、3.65 和 3.23 倍。该方法将有助于操纵涉及灵芝中多个基因的复杂代谢或调控途径。
